THE  ROLE  OF  SALTS  IN  THE  PRESERVATION 

OF  LIFE 


-f 


JACQUES  LOEB 


[ Reprinted  from  Science,  N.  S.,  Vol.  XXXIV.,  No.  881 , Pages  658-665,  November  17,  1911 ] 


•.  517A 

Demote  ^ 

[Reprinted  from  Science,  iV,  Vol.  XXXIV.,  No.  881 , Pages  653-665 , November  17 , 191  i] 

. £ 


THE  ROLE  OF  SALTS  IN  THE  PRESERVATION  OF  LIFE 


Less  is  known  of  the  role  of  the  salts  in 
the  animal  body  than  of  the  role  of  the 
three  other  main  food-stuffs,  namely,  carbo- 
hydrates, fats  and  proteins.  As  far  as  the 
latter  are  concerned,  we  know  at  least  that 
through  oxidation  they  are  capable  of 
furnishing  heat  and  other  forms  of  energy. 
The  neutral  salts,  * however,  are  not  oxi- 
dizable.  Yet  it  seems  to  be  a fact  that  no 
animal  can  live  on  an  ash-free  diet  for  any 
length  of  time,  although  no  one  can  say 
why  this  should  be  so.  We  have  a point  of 
attack  for  the  investigation  of  the  role  of 
the  salts  in  the  fact  that  the  cells  of  our 
body  live  longest  in  a liquid  which  con- 
tains the  three  salts,  NaCl,  KC1  and  CaCl2 
x in  a definite  proportion,  namely,  100  mole- 
^ cules  NaCl,  2.2  molecules  KC1  and  1.5 
molecules  of  CaCl2.  This  proportion  is 
t)  identical  with  the  proportion  in  which 
£ these  salts  are  contained  in  sea- water;  but 
<4_  the  concentration  of  the  three  salts  is  not 
0 the  same  in  both  cases.  It  is  about  three 
times  as  high  in  the  sea-water  as  in  our 
^ blood  serum. 

O Biologists  have  long  been  aware  of  the 
c fact  that  the  ocean  has  an  incomparably 
>^richer  fauna  than  fresh-water  lakes  or 
^ streams  and  it  is  often  assumed  that  life  on 
rt~our  planet  originated  in  the  ocean.  The 
~Z  fact  that  the  salts  of  Na,  Ca  and  Kf  exist 

«n 

04  1 Carpenter  lecture  delivered  at  the  Academy  of 

Medicine  of  New  York,  October  39,  1911. 


in  the  same  proportion  in  our  blood  serum 
as  in  the  ocean  has  led  some  authors  to  the 
conclusion  that  our  ancestors  were  marine 
animals,  and  that,  as  a kind  of  inheritance, 
we  still  carry  diluted  sea-water  in  our 
blood.  Statements  of  this  kind  have  mainly 
a metaphorical  value,  but  they  serve  to 
emphasize  the  two  facts,  that  the  three 
salts,  NaCl,  KC1  and  CaCl2,  exist  in  our 
blood  in  the  same  relative  proportion  as  in 
the  ocean  and  that  they  seem  to  play  an 
important  role  in  the  maintenance  of  life. 

I intend  to  put  before  you  a series  of 
experiments  which  seem  to  throw  some 
light  on  the  mechanism  by  which  the  solu- 
tions surrounding  living  cells  influence 
their  duration  of  life. 

n 

In  order  to  give  a picture  of  the  extent 
to  which  the  life  of  many  animals  depends 
upon  the  cooperation  of  the  three  salts  I 
may  mention  experiments  made  on  a small 
marine  crustacean,  Gammarus,  of  the  Bay 
of  San  Francisco.  If  these  animals  are 
suddenly  thrown  into  distilled  water,  their 
respiration  stops  (at  a temperature  of 
20°  C.)  in  about  half  an  hour.  If  they  are 
put  back  immediately  after  the  cessation 
of  respiration  into  sea-water,  they  can  re- 
cuperate. If  ten  minutes  or  more  are  al- 
lowed to  elapse  before  bringing  them  back 
into  the  sea-water,  no  recuperation  is  pos- 
sible. Since  in  this  case  death  is  caused 


2 


SCIENCE 


obviously  through  the  entrance  of  distilled 
water  into  the  tissues  of  the  animals,  one 
would  expect  that  the  deadly  effect  of  dis- 
tilled water  would  be  inhibited  if  enough 
cane  sugar  were  added  to  the  distilled 
water  to  make  the  osmotic  pressure  of  the 
solution  equal  to  that  of  the  sea-water.  If, 
however,  the  animals  are  put  into  cane- 
sugar  solution,  the  osmotic  pressure  of 
which  is  equal  to  that  of  sea-water,  the 
animals  die  just  about  as  rapidly  as  in  dis- 
tilled water.  The  same  is  true  if  the  os- 
motic pressure  of  the  sugar  solution  is 
higher  or  lower  than  that  of  the  sea-water. 
The  sugar  solution  is,  therefore,  about  as 
toxic  for  the  animals  as  the  distilled  water, 
although  in  the  latter  case  water  enters 
into  the  tissues  of  the  animal,  while  in  the 
former  case  it  does  not. 

If  the  sea-water  is  diluted  with  an 
equal  quantity  of  distilled  water  in  one 
case,  and  of  isotonic  cane-sugar  solution 
in  the  other  case,  in  both  cases  the  dura- 
tion of  life  is  shortened  by  practically  the 
same  amount. 

If  the  crustaceans  are  brought  into  a 
pure  solution  of  NaCl,  of  the  same  osmotic 
pressure  as  the  sea-water,  they  also  die  in 
about  half  an  hour.  If  to  this  solution  a 
little  calcium  chloride  be  added  in  the 
proportion  in  which  it  is  contained  in  the 
sea-water  the  animals  die  as  rapidly  as 
without  it.  If,  however,  both  CaCl2  and 
KC1  are  added  to  the  sodium  chloride  so- 
lution, the  animals  can  live  for  several 
days.  The  addition  of  KC1  alone  to  the 
NaCl  prolongs  their  life  but  little. 

If  KC1  and  CaCl2  are  added  to  a cane 
sugar  solution  isotonic  with  sea-water,  the 
animals  die  as  quickly  or  more  so  than  in 
the  pure  cane-sugar  solution. 

If  other  salts  be  substituted  for  the  three 
salts  the  animals  die.  The  only  substitu- 
tion possible  is  that  of  SrCl2  for  CaCl2. 
We  find  also  that  the  proportion  in  which 
the  three  salts  of  sodium,  calcium  and 


potassium  have  to  exist  in  the  solution 
can  not  be  altered  to  any  extent.  All  this 
leads  us  to  the  conclusion,  that  in  order  to 
preserve  the  life  of  the  crustacean  Gam- 
marus,  the  solution  must  not  only  have  a 
definite  concentration  or  osmotic  pressure 
but  that  this  osmotic  pressure  must  be 
furnished  by  definite  salts,  namely,  sodium 
chloride,  calcium  chloride  and  potassium 
chloride  in  the  proportion  in  which  these 
three  salts  exist  in  the  sea-water  (and  in 
the  blood) ; this  fact  could  also  be  demon- 
strated for  many  other  marine  animals. 
The  relative  tolerance  of  various  cells  and 
animals  for  abnormal  salt  solutions  is, 
however,  not  the  same,  a point  which  we 
shall  discuss  later  on. 

iii 

What  is  the  role  of.  the  salts  in  these 
cases?  The  botanists  have  always  consid- 
ered salt  solutions  as  nutritive  solutions. 
It  is  a well-known  fact  that  plants  require 
definite  salts,  e.  g.,  nitrates  and  potassium 
salts,  for  their  nutrition,  and  the  question 
now  arises  whether  the  three  salts  NaCl, 
KC1  and  CaCl2,  which  are  needed  for  the 
preservation  of  animal  life,  play  the  role 
of  nutritive  salts.  Experiments  which  I 
made  on  a small  marine  fish,  Fundulus , 
proved  beyond  question  that  this  is  not  the 
case.  If  the  young,  newly  hatched  fish  are 
put  into  a pure  solution  of  sodium  chloride 
of  the  concentration  in  which  this  salt  is 
contained  in  sea-water,  the  animals  very 
soon  die.  If,  however,  KC1  and  CaCl2  be 
added  to  the  solution  in  the  right  proportion, 
the  animals  can  live  indefinitely.  These  fish, 
therefore,  behave  in  this  respect  like  Gam- 
marus  and  the  tissues  of  the  higher  ani- 
mals, but  they  differ  from  Gammarus  and 
the  majority  of  marine  animals  inasmuch 
as  the  fish  can  live  long,  and  in  some  cases, 
indefinitely,  in  distilled  and  fresh  water, 
and  certainly  in  a very  dilute  solution  of 
sodium  chloride.  From  this  fact  I drew 


SCIENCE 


3 


the  conclusion  that  KC1  and  CaCl2  do  not 
act  as  nutritive  substances  for  these  ani- 
mals, that  they  only  serve  to  render  NaCl 
harmless  if  the  concentration  of  the  latter 
salt  is  too  high.  I succeeded  in  showing  that 
as  long  as  the  sodium-chloride  solution  is 
very  dilute  and  does  not  exceed  the  con- 
centration of  m./8,  the  addition  of  KC1 
and  CaCl2  is  not  required.  Only  when  the 
solution  of  NaCl  has  a concentration  above 
m./8  does  it  become  harmful  and  does  it 
require  the  addition  of  KC1  and  CaCl2. 

The  experiments  on  Fundulus,  therefore, 
prove  that  a mixture  of  NaCl  + KC1  + 
CaCl2  does  not  act  as  a nutritive  solution, 
but  as  a 'protective  solution.  KC1  and 
CaCl2  are  only  necessary  in  order  to  pre- 
vent the  harmful  effects  which  NaCl  pro- 
duces if  it  is  alone  in  solution  and  if  its 
concentration  is  too  high.  We  are  dealing, 
in  other  words,  with  a case  of  antagonistic 
salt  action;  an  antagonism  between  NaCl 
on  the  one  hand  and  KC1  and  CaCl2  on  the 
other.  The  discovery  of  antagonistic  salt 
action  was  made  by  Ringer,  who  found  that 
there  is  a certain  antagonism  between  K 
and  Ca  in  the  action  of  the  heart.  When 
he  put  the  heart  of  a frog  into  a mixture 
of  NaCl  + KC1  he  found  that  the  contrac- 
tions of  the  heart  were  not  normal,  but  they 
were  rendered  normal  by  the  addition  of  a 
little  CaCl2.  A mixture  of  NaCl  + CaCl2 
also  caused  abnormal  contractions  of  the 
heart,  but  these  were  rendered  normal  by 
the  addition  of  KC1.  Ringer  drew  the 
conclusion  that  there  existed  an  antagonism 
between  potassium  and  calcium,  similar  to 
that  which  Schmiedeberg  had  found  be- 
tween different  heart  poisons,  e.  g.,  atropin 
and  muscarin.  Biedermann  had  found 
that  alkaline  salt  solutions  cause  twitchings 
in  the  muscle  and  Ringer  found  that  the 
addition  of  Ca  inhibited  these  twitchings. 
Since  these  experiments  were  made  many 
examples  of  the  antagonistic  action  of  salts 
have  become  known. 


It  had  generally  been  assumed  that  the 
antagonistic  action  of  two  salts  was  based 
on  the  fact  that  each  salt,  when  applied 
singly,  acted  in  the  opposite  way  from  that 
of  its  antagonist.  We  shall  see  that  in  cer- 
tain cases  of  antagonistic  salt  action  at 
least  this  view  is  not  supported  by  fact. 

IV 

What  is  the  mechanism  of  antagonistic 
salt  action?  I believe  that  an  answer  to 
this  question  lies  in  the  following  observa- 
tions on  the  eggs  of  Fundulus.  If  these 
eggs  are  put  immediately  after  fertilization 
into  a pure  sodium  chloride  solution  which 
is  isotonic  with  sea-water,  they  usually  die 
without  forming  an  embryo.  If,  however, 
only  a trace  of  a calcium  salt,  or  of  any 
other  salt  with  a bivalent  metal  (with  the 
exception  of  Hg,  Cu  or  Ag)  is  added  to 
the  m./2  NaCl  solution,  the  toxicity  of  the 
solution  is  diminished  or  even  abolished. 
Even  salts  which  are  very  poisonous, 
namely,  salts  of  Ba,  Zn,  Pb,  Ko,  Ni,  Mn 
and  other  bivalent  metals,  are  able  to  ren- 
der the  pure  solution  of  sodium  chloride 
harmless,  at  least  to  the  extent  that  the 
eggs  can  live  long  enough  to  form  an  em- 
bryo. The  fact  that  a substance  as  poison- 
ous as  Zn  or  lead  can  render  harmless  a 
substance  as  indifferent  as  sodium  chloride 
seemed  so  paradoxical  that  it  demanded  an 
explanation,  and  this  explanation  casts  light 
on  the  nature  of  the  protective  or  antagon- 
istic action  of  salts.  For  the  antagonistic 
action  of  a salt  of  lead  or  zinc  against  the 
toxic  action  of  sodium  chloride  can  only 
consists  in  the  lead  salt  protecting  the  em- 
bryo against  the  toxic  action  of  the  NaCl. 
But  how  is  this  protective  action  possible? 

We  have  mentioned  that  if  we  put  the 
young  fish,  immediately  after  hatching,  into 
a pure  m./2  solution  of  sodium  chloride 
the  animals  die  very  quickly,  but  that  they 
live  indefinitely  in  the  sodium  chloride  solu- 
tion if  we  add  both  CaCl2  and  KC1.  Plow 


4 


SCIENCE 


does  it  happen  that  for  the  embryo,  as  long 
as  it  is  in  the  egg  shell,  the  addition  of 
CaCl2  to  the  NaCl  solution  suffices,  while 
if  the  fish  is  out  of  the  shell  the  addition 
of  CaCl2  alone  is  no  longer  sufficient  and 
the  addition  of  KC1  also  becomes  neces- 
sary? Moreover,  if  we  try  to  preserve  the 
life  of  the  fish  after  it  is  taken  out  of  the 
egg  in  an  m./2  sodium  chloride  solution  by 
adding  ZnS04,  or  lead  acetate,  to  the  solu- 
tion we  find  that  the  fish  die  even  much 
more  quickly  than  without  the  addition. 

If  we  look  for  the  cause  of  this  difference 
our  attention  is  called  to  the  fact  that  the 
fish,  as  long  as  it  is  in  the  egg,  is  separated 
from  the  surrounding  solution  by  the  egg 
membrane.  This  egg  membrane  possesses 
a small  opening,  the  so-called  micropyle, 
through  which  the  spermatozoon  enters  into 
the  egg.  I have  gained  the  impression  that 
this  micropyle  is  not  closed  as  tightly  im- 
mediately after  fertilization  as  later  on, 
since  the  newly  fertilized  egg  is  killed  more 
rapidly  by  an  m./2  solution  of  NaCl  than 
it  is  killed  by  the  same  solution  one  or  two 
days  after  fertilization.  One  can  imagine 
that  the  micropyle  contains  a wad  of  a col- 
loidal substance  which  is  hardened  gradu- 
ally to  a leathery  consistency  if  the  egg 
remains  in  the  sea-water.  With  the  proc- 
ess of  hardening,  or  tanning,  it  becomes 
more  impermeable  for  the  NaCl  solution. 
This  process  of  hardening  is  brought  about 
apparently  very  rapidly  if  we  add  to  the 
m./2  NaCL  solution  a trace  of  a salt  of  a 
bivalent  metal  like  Ca,  Sr,  Ba,  Zn,  Pb,  Mn, 
Ko  and  Ni,  etc.  It  is  also  possible  that 
similar  changes  take  place  in  the  whole 
membrane.  The  process  of  rendering  the 
m./2  Na  solution  harmless  for  the  embryo 
of  the  fish,  therefore,  depends  apparently 
upon  the  fact  that  the  addition  of  the  bi- 
valent metals  renders  the  micropyle  or  per- 
haps the  whole  membrane  of  the  egg  more 
impermeable  to  NaCl  than  was  the  case 
before. 


But  these  are  only  one  part  of  the  facts 
which  throw  a light  upon  the  protective  or 
antagonistic  action  of  salts.  Further  data 
are  furnished  by  experiments  which  I made 
together  with  Professor  Gies,  also  on  the 
eggs  of  Fundulus.  Gies  and  I were  able  to 
show  that  not  only  are  the  bivalent  metals 
able  to  render  the  sodium  chloride  solution 
harmless,  but  that  the  reverse  is  also  the 
case,  namely,  that  NaCl  is  required  to 
render  the  solutions  of  many  of  the  bi- 
valent metals,  for  instance  ZnS04,  harm- 
less. (That  the  S04  ion  has  nothing  to  do 
with  the  result  was  shown  before  by  experi- 
ments with  Na2S04.) 

TABLE  I 

Percentage  of 
the  Eggs  Form- 


Nature  of  the  Solution  ing  an  Embryo 

100  c.c.  distilled  water  49 

100  c.c.  distilled  water 

+ 8 c.c.  m./32  ZnS04  0 

100  c.c.  m./64  NaCl+8  c.c.  m./32  ZnS04  0 

100  c.c.  m./32  NaCl+8  c.c.  m./32  ZnS04  3 

100  c.c.  m./16  NaCl+8  c.c.  m./32  ZnS04  8 

100  c.c.  m./8  NaCl+8  c.c.  m./32  ZnS04  44 

100  c.c.  m./4  NaCl+8  c.c.  m./32  ZnS04  38 

100  c.c.  3/8  NaCl+8  c.c.  m./32  ZnS04  37 

100  c.c.  m./2  NaCl+8  c.c.  m./32  ZnS04  34 

100  c.c.  5/8  NaCl+8  c.c.  m./32  ZnS04  29 

100  c.c.  6/8  NaCl+8  c.c.  m./32  ZnS04  8 

100  c.c.  7/8  NaCl+8  c.c.  m./32  ZnS04  6 

100  c.c.  m.  NaCl+8  c.c.  m./32  ZnS04  1 


If  the  eggs  of  Fundulus  are  put  imme- 
diately after  fertilization  into  distilled 
water,  a large  percentage  of  the  eggs  de- 
velop, often  as  many  as  one  hundred  per 
cent.,  and  the  larvae  and  embryos  formed 
in  the  distilled  water  are  able  to  hatch. 
If  we  add,  however,  to  100  c.c.  of  distilled 
water  that  quantity  of  ZnS04  which  is  re- 
quired to  render  the  NaCl  solution  harm- 
less, all  the  eggs  are  killed  rapidly  and  not 
a single  one  is  able  to  form  an  embryo. 
If  we  add  varying  amounts  of  NaCl  we  find 
that,  beginning  with  a certain  concentration 
of  NaCl,  this  salt  inhibits  the  toxic  effects 
of  ZnS04  and  many  eggs  are  able  to  form 


SCIENCE 


5 


an  embryo.  This  can  be  illustrated  by 
Table  I. 

This  table  shows  that  the  addition  of 
NaCI,  if  its  concentration  exceeds  a certain 
limit,  namely,  m./8,  is  able  to  render  the 
ZnS04  in  the  solution  comparatively  harm- 
less. 

If  we  now  assume  that  ZnS04  renders 
the  5/8  m.  NaCI  solution  harmless  by  ren- 
dering the  egg  membrane  comparatively 
impermeable  for  NaCI  we  must  also  draw 
the  opposite  conclusion,  namely,  that  NaCI 
renders  the  egg  membrane  comparatively 
impermeable  for  ZnS04.  We,  therefore, 
arrive  at  a new  conception  of  the  mutual 
antagonism  of  two  salts,  namely,  that  this 
antagonism  depends,  in  this  case  at  least, 
upon  a common,  cooperative  action  of  both 
salts  on  the  egg  membrane,  by  which  action 
this  membrane  becomes  completely  or  com- 
paratively impermeable  for  both  salts. 
And  from  this  we  must  draw  the  further 
conclusion  that  the  fact  that  each  of  these 
salts,  if  it  is  alone  in  the  solution,  is  toxic, 
is  due  to  its  comparatively  rapid  diffusion 
through  the  membrane,  so  that  it  comes 
into  direct  contact  with  the  protoplasm  of 
the  germ. 

As  long  as  we  assumed  that  each  of  the 
two  antagonistic  salts  s acted,  if  applied 
singly,  in  the  opposite  way  from  its  antag- 
onist, it  was  impossible  to  understand  these 
experiments  or  find  an  analogue  for  them 
in  colloid  chemistry.  But  if  we  realize 
that  NaCI  alone  is  toxic  because  it  is  not 
able  to  render  the  egg  membrane  imper- 
meable ; and  that  ZnS04  if  alone  in  solution 
is  toxic  for  the  same  reason;  while  both 
combined  are  harmless  (since  for  the  “tan- 
ning” of  the  membrane  the  action  of  the 
two  salts  is  required)  these  experiments 
become  clear. 

We  may,  for  the  sake  of  completeness, 
still  mention  that  salts  alone  have  such 
antagonistic  effects ; glycerine,  urea  and 
alcohol  have  no  such  action.  On  the  other 


hand,  ZnS04  was  not  only  able  to  render 
NaCI  harmless,  but  also  LiCl,  NH4C1,  CaCl2 
and  others ; and  vice  versa. 

These  experiments  on  the  egg  of  Fun- 
dulus  are  theoretically  of  importance,  since 
they  leave  no  doubt  that  in  this  case  at 
least  the  ‘ ‘ antagonistic  ’ ’ action  of  salts  con- 
sists in  a modification  of  the  egg  membrane 
by  a combined  action  of  two  salts,  whereby 
the  membrane  becomes  less  permeable  for 
both  salts. 

v 

It  is  not  easy  to  find  examples  of  experi- 
ments in  the  literature  which  are  equally 
unequivocal  in  regard  to  the  character  of 
antagonistic  salt  action;  but  I think  that 
some  recent  experiments  by  Osterhout  sat- 
isfy this  demand. 

It  has  long  been  a question  whether  or 
not  cells  are  at  all  permeable  for  salts. 
Nobody  denies  that  salts  diffuse  much  more 
slowly  into  the  cells  than  water;  but  some 
authors,  especially  Overton  and  IToeber, 
deny  categorically  that  salts  can  diffuse  at 
all  into  the  cells.  Overton’s  view  is  based 
partly  on  experiments  on  plasmolysis  in 
the  cells  of  plants.  If  the  cells  of  plants, 
for  example,  those  of  Spirogyra,  are  put 
into  a solution  of  NaCI  or  some  other  salt 
of  sufficiently  high  osmotic  pressure,  the 
volume  of  the  contents  of  the  cell  decreases 
through  loss  of  water  and  the  protoplasm 
retracts,  especially  from  corners  of  the 
rigid  cellulose  walls.  Overton  maintains 
that  this  plasmolysis  is  permanent,  and  con- 
cludes from  this  that  only  water  but  no 
salt,  can  diffuse  through  the  cell- wall ; since 
otherwise  salts  should  gradually  diffuse 
from  the  solution  into  the  cell,  and  through 
this  increase  in  the  osmotic  pressure  of  the 
cell  the  water  should  finally  diffuse  back 
into  the  cell  and  restitute  the  normal  vol- 
ume of  the  cell.  According  to  Overton  this 
does  not  happen. 

Osterhout  has  recently  shown  that  Over- 


6 


SCIENCE 


ton’s  observations  were  incomplete  in  a 
very  essential  point  and  that  in  reality  the 
plasmolysis,  which  occurs  in  this  case  when 
the  cell  is  put  into  the  hypertonic  solution, 
disappears  again  in  a time  which  varies 
with  the  nature  of  the  salt  in  solution. 
This  stage  of  reversion  of  plasmolysis  had 
been  overlooked  by  Overton.  If  the  cell, 
however,  remains  permanently  in  the  hy- 
pertonic sodium  chloride  solution,  after- 
wards again  a shrinking  of  the  contents  of 
the  cell  takes  place,  which  superficially  re- 
sembles plasmolysis,  but  which  in  reality 
has  nothing  to  do  with  plasmolysis,  but  is  a 
phenomenon  of  death.  That  this  second 
“false  plasmolysis,”  as  Osterhout  calls  it, 
has  nothing  to  do  with  the  hypertonic  char- 
acter of  the  solution  was  proved  by  the  fact 
that  hypotonic  solutions  of  toxic  substances 
may  produce  the  same  phenomenon. 

In  one  experiment  which  Osterhout  de- 
scribes, “a  portion  of  a Spirogyra  filament 
was  plasmolyzed  in  .2  m.  CaCl2,  but  not  in 
.195  m.  CaCl2.  A .29  m.  NaCl  solution  has 
approximately  the  same  osmotic  pressure  as 
d .2  m.  CaCl2  solution.  But  on  placing 
another  portion  of  the  same  Spirogyra  fila- 
ment in  a .29  m.  NaCl  solution  the  expected 
plasmolysis  does  not  occur  and  it  is  impos- 
sible to  plasmolyze  the  cells  until  they  are 
placed  in  .4  m.  NaCl.  ’ ’ Osterhout  explains 
this  difference  in  the  concentration  of  the 
two  salts  required  for  plasmolysis  by  the 
assumption  that  NaCl  diffuses  more  rapidly 
into  the  cell  than  CaCl2,  a conclusion  which 
I reached  also  on  the  basis  of  my  earlier 
experiments  on  animals. 

Osterhout ’s  experiments  also  show  that 
the  antagonism  of  NaCl  and  CaCl2  depends 
partly  on  the  facts  that  the  two  salts  in- 
hibit each  other  from  diffusing  into  the 
cells,  and  this  conclusion  is  based  among 
others  upon  the  following  experiment. 
“By  dividing  a Spirogyra  filament  into 
several  portions  it  was  found  that  it  was 
plasmolyzed  in  .2  m.  CaCl2  and  in  .38  m. 


NaCl,  but  neither  in  .195  m.  CaCl2  nor  in 
.375  m.  NaCl.  On  mixing  100  c.c.  .375  m. 
NaCl  with  10  c.c.  .195  m.  CaCl2  and  placing 
other  portions  of  the  same  filament  in  it, 
prompt  and  very  marked  plasmolysis  oc- 
curred. ’ ’ 

The  explanation  for  this  observation  lies 
in  the  fact  that  in  the  mixture  of  NaCl  and 
CaCl2  the  two  salts  render  their  diffusion 
into  the  cell  mutually  more  difficult.  After 
a longer  period  of  time  the  plasmolyzed 
cells  can  expand  again  in  a mixture  of 
NaCl  and  CaCl2,  but  that  occurs  much  later 
than  if  they  are  in  the  pure  NaCl  solution. 

These  experiments  are  the  analogue  of 
the  observation  on  the  embryo  of  the  eggs 
of  Fundulus  in  which  a pure  solution  of 
ZnS04  diffused  rapidly  through  the  mem- 
brane or  micropyle,  while,  if  both  salts  were 
present,  the  diffusion  was  inhibited  or  con- 
siderably retarded. 

While  the  observations  of  Osterhout  show 
that  Overton  was  not  justified  in  using  the 
experiments  on  plasmolysis  to  prove  that 
the  neutral  salts  can  not  diffuse  into  the 
cells,  yet  they  do  not  prove  that  these  salts 
diffuse  into  the  cell  under  normal  condi- 
tions. In  Osterhout ’s  experiments  the  cells 
are  in  strongly  hypertonic  solutions  and  it 
does  not  follow  that  such  solutions  act  like 
isotonic,  perfectly  balanced  solutions. 

VI 

Wasteneys  and  I have  recently  shown 
that  the  toxic  action  of  acids  upon  Fun- 
dulus can  be  annihilated  by  salts.  If  we 
add  0.5  c.c.  A/10  butyric  acid  to  100  c.c.  of 
distilled  water  these  fish  die  in  2J  hours  or 
less.  In  solutions  which  contain  0.4  c.c.  or 
less  acid  they  can  live  for  a week  or  more. 
If  we  add,  however,  0.5  c.c.  of  butyric  acid 
to  100  c.c.  of  solutions  of  NaCl  of  various 
concentration,  we  find  that  above  a certain 
limit  the  NaCl  can  render  the  acid  harm- 
less. It  is  needless  to  say  that  the  NaCl 
used  in  these  experiments  was  strictly  neu- 


SCIENCE 


7 


tral  and  that  the  amount  of  acid  present  in 
the  mixture  of  acid  and  salt  was  measured. 
The  following  experiment  may  serve  as  an 
example.  Each  solution  contained  six  fish 
at  the  beginning  of  the  experiment. 


TABLE  II 


After 

Number  of  Surviving  Fish  in  0.5  c.c. 
Butyric  Acid 

W/10 

+°  | 

4.0  | 

6.0  | 

8.0  | 

10.0 

12.0 

15  0 

c.c. 

. m./2  NaCl  in  100  c.c.  of  the  Solution 

2 hours 

0 

0 

0 

2 

3 

3 

6 

4 hours  

0 

3 

2 

5 

1 day 

1 

1 

5 

2 days 

1 

0 

5 

3 days 

1 

5 

4 days 

1 

5 

If  the  amount  of  acid  was  increased,  the 
amount  of  NaCl  also  had  to  be  increased  to 
render  the  acid  harmless.  In  order  to  ren- 
der 0.5  c.c.  iV/lO  butyric  acid  pro  100  c.c. 
solution  harmless,  10  c.c.  m./2  NaCl  had  to 
be  added;  while  0.8  c.c.  butyric  acid  re- 
quired 20  c.c.  and  1.0  c.c.  butyric  acid  re- 
quired about  28  c.c.  m./2  NaCl  in  100  c.c. 
of  the  solution. 

Not  only  butyric  acid,  but  any  kind  of 
acid,  could  be  rendered  harmless  by  neutral 
salts,  e.  g.,  HC1  by  NaCl. 

It  is  of  great  importance  that  the  an- 
tagonistic action  of  CaCl2  was  found  to  be 
from  8 to  11  times  as  great  or  powerful  as 
the  action  of  NaCl.  This  harmonizes  with 
the  general  observation  that  the  protective 
action  of  CaCl2  for  the  life  of  cells  is 
greater  than  that  of  any  other  substance. 

Wasteneys  and  I could  show  that  the  rate 
of  the  absorption  of  acid  by  the  fish  is  the 
same  in  solutions  with  and  without  salt. 
This  proves  that  the  action  of  the  salts  con- 
sisted in  this  case  not  in  preventing  the 
diffusion  or  absorption  of  the  acid,  but  in 
modifying  the  deleterious  effect  of  the  ab- 
sorbed acid. 

We  can  state  a little  more  definitely  the 
cause  of  death  by  acid.  If  we  put  the  fish 
into  a weak  acid  solution  in  distilled  water 


just  strong  enough  to  kill  the  fish  in  from 
1 to  2 hours  ( e . g.,  500  c.c.  H20  + 2.0  c.c. 
iV/10  HC1),  we  notice  that  the  acid  very 
soon  makes  the  normally  transparent  epi- 
dermis of  the  fish  opaque,  and  a little  later 
the  epidermis  falls  off  in  pieces  and  shreds. 
This,  however,  is  probably  not  the  direct 
cause  of  the  death,  but  I am  inclined  to 
assume  that  the  fish  die  from  suffocation 
caused  by  a similar  action  of  the  acid  upon 
the  gills. 

The  action  of  the  acid  upon  the  epidermis 
of  the  body  as  well  as  upon  the  gills  is  pre- 
vented through  the  addition  of  neutral 
salts. 

It  is  well  known  that  the  action  of  acids 
upon  proteins  can  be  inhibited  by  neutral 
salts.  Thus  the  internal  friction  of  certain 
protein  solutions  is  increased  by  acids  while 
the  addition  of  neutral  salts  inhibits  this 
effect  (Pauli).  The  swelling  of  gelatine 
caused  by  acid  is  inhibited  by  salts 
(Procter). 

It  is  possible  that  in  the  experiments 
with  acid  the  fish  is  killed  in  the  following 
way.  The  acid  causes  certain  proteins  in 
the  surface  layer  of  the  epithelial  cells  of 
the  gills  and  of  the  skin  to  swell,  whereby 
this  surface  layer  becomes  more  permeable 
for  the  acid.  The  acid  can  now  diffuse  into 
the  epithelial  cells  and  act  on  the  proto- 
plasm, whereby  the  cells  are  killed.  If 
salts  are  present  in  the  right  concentra- 
tion, the  combined  action  of  acid  and  salt 
causes  a dehydration  of  the  surface  film  of 
these  cells,  as  it  does  in  the  experiments  on 
gelatine  or  as  in  the  cases  of  tanning  of 
hides  by  the  combined  action  of  acids  and 
salt  solutions.  This  combined  dehydrating 
or  “ tanning”  action  of  acid  and  salts  on 
the  surface  of  the  epithelial  cells  of  the 
gills  diminishes  the  permeability  of  this 
layer  for  the  acids  and  prevents  them  from 
diffusing  into  the  cells  and  thus  destroying 
the  protoplasm.  In  this  way  the  gills  are 
kept  intact  and  the  life  of  the  fish  is  saved. 


8 


SCIENCE 


As  long  as  the  amount  of  acid  is  small 
the  amount  absorbed  is  not  essentially 
diminished  by  the  presence  of  salts;  but 
while  in  the  presence  of  salts  the  acid  is 
consumed  in  the  tanning  action  of  the  sur- 
face layer  of  the  cells,  or  is  absorbed  in 
this  layer ; if  no  salt  is  present  part  of  the 
acid  diffuses  into  the  epithelial  cells  and 
kills  the  latter. 

VII 

We  have  thus  far  considered  the  cases  of 
antagonism  between  two  electrolytes  only. 
The  case  of  the  antagonism  between  three 
electrolytes  is  a little  more  complicated. 

We  choose  as  an  example  the  antagonism 
between  NaCl,  KC1  and  CaCl2 — the  antag- 
onism which  is  most  important  in  life  phe- 
nomena. If  the  mechanism  of  the  antag- 
onism between  NaCl,  on  the  one  hand,  and 
KC1  and  CaCl2,  on  the  other,  is  of  the  same 
nature  as  that  between  NaCl  and  ZnS04  in 
the  case  of  the  eggs  of  Fundulus,  it  must 
be  possible  to  show  that  not  only  is  NaCl 
toxic  if  it  is  alone  in  solution,  and  that  it  is 
rendered  harmless  by  the  two  other  salts, 
but  that  the  reverse  is  true  also.  This  can 
be  proved  in  the  case  of  KC1.  To  demon- 
strate it,  we  have  again  to  experiment  on 
organisms  which  are,  in  wide  limits,  inde- 
pendent of  the  osmotic  pressure  of  the  sur- 
rounding solution  since  the  concentration 
of  the  KC1  in  sea-water  is  very  low.  The 
experiments  were  carried  out  by  Mr.  Was- 
teneys  and  myself  on  Fundulus . The 
method  consisted  in  putting  six  fish,  after 
washing  them  twice  with  distilled  water, 
into  500  c.c.  of  the  solution.  It  was  ascer- 
tained from  day  to  day  how  many  fish 
survived. 

When  the  fish  were  put  into  pure  solu- 
tions of  KC1  of  the  concentration  in  which 
this  salt  is  contained  in  the  sea-water 
(2.2  c.c.  m./2  KC1  in  100  c.c.  of  the  solu- 
tion) they  died  mostly  in  less  than  two 
days.  This  is  not  due  to  the  low  concentra- 
tion of  the  KC1  solution,  which  is  only  1/50 


of  that  of  the  sea-water,  since  the  fish  can 
live  indefinitely  in  a pure  NaCl  solution  of 
the  same  concentration  as  that  in  which  the 
KC1  exists  in  the  sea-water. 

If  we  add  to  the  toxic  quantities  of  KC1 
increasing  quantities  of  NaCl,  we  find  that 
as  soon  as  the  solution  contains  17  or  more 
molecules  of  NaCl  to  one  molecule  of  KC1, 
the  toxic  action  of  KC1  is  considerably 
diminished,  if  not  completely  counteracted. 
The  following  table  may  serve  as  an  ex- 
ample. 

TABLE  III 


More  accurate  determinations  showed 
that  already  a 3/16  m.  NaCl  solution  ren- 
ders the  solution  of  2.2  c.c.  m./2  KC1  in 
100  c.c.  of  the  solution  harmless. 

TABLE  IV 

Coefficient  of 
Antagonization 


0.6  c.c.  m./2  KC1  rendered  harmless  in 

100  c.c.  3/64  m.  NaCl  1/16 

0.7  c.c.  m./2  KC1  rendered  harmless  in 

100  c.c.  4/64  m.  NaCl  1/18 

0.9  c.c.  m./2  KC1  rendered  harmless  in 

100  c.c.  5/64  m.  NaCl  1/17 

1.0  c.c.  m./2  KC1  rendered  harmless  in 

100  c.c.  5/64-6/64  m.  NaCl  ....  1/16-1/19 

1.1  c.c.  m./2  KC1  rendered  harmless  in 

100  c.c.  6/64  m.  NaCl 1/17 

1.65  c.c.  m./2  KC1  rendered  harmless  in 

100  c.c.  5/32  m.  NaCl  1/19 

2.2  c.c.  m./2  KC1  rendered  harmless  in 

100  c.c.  6/32  m.  NaCl  1/17 

2.75  c.c.  m./2  KC1  rendered  harmless  in 

100  c.c.  7/32  m.  NaCl  1/16 

3.3  c.c.  m./2  KC1  rendered  harmless  in 

100  c.c.  9/32  m.  NaCl 1/17 


It  was  next  determined  whether  different 
concentrations  of  KC1  required  different 


SCIENCE 


9 


concentrations  of  NaCl.  It  was  found  that 
the  coefficient  of  antagonization  KCl/NaCl 
has  an  approximately  constant  value, 
namely,  about  1/17,  as  Table  IV.  shows. 

What  happens  if  we  vary  this  ratio  ? If 
we  add  too  little  NaCl  to  the  KC1  solution, 
namely,  only  1 to  10  molecules  NaCl  to 
1 molecule  of  KC1,  the  solution  becomes 
more  harmful  than  if  KC1  is  alone  in  solu- 
tion; if  we  add  considerably  more  than  17 
molecules  NaCl,  e.  g.,  50  molecules  to  one 
molecule  of  KC1,  the  solution  becomes  toxic 
again ; and  the  more  so  the  higher  the  con- 
centration of  NaCl.  This  indicates  that 
the  antagonistic  effect  requires  a rather 
definite  ratio  of  the  two  salts.  This  fur- 
nishes the  reason  why  an  m./2  solution  can, 
as  a rule,  not  be  rendered  completely  harm- 
less by  the  mere  addition  of  KC1,  but  that 
in  addition  CaCl2  is  needed. 

If  we  add  to  100  c.c.  m./2  NaCl  enough 
KC1  to  make  the  ratio  KC1 : NaCl  = 1/17 
we  find  that  the  antagonization  of  KC1: 
NaCl  becomes  incomplete.  If  the  amount 
of  KC1  in  100  c.c.  of  the  solution  exceeds 
2.2  c.c.  m./2  KC1,  antagonization  is  still  to 
some  extent  possible,  but  it  becomes  more 
incomplete  the  higher  the  concentration  of 
KC1.  For  this  reason  it  is  not  possible  to 
render  an  m./2  solution  of  NaCl  harmless 
by  the  mere  addition  of  KC1. 

TABLE  V 

Coefficient  of  Antago- 
nization KCl/0aCl2 


1.1  c.c.  m./2  KC1  in  100  c.c.  H20 

require  0.1  m./lOO  CaCLj  . . 550 

1.65  c.c.  m./2  KC1  in  100  c.c.  H20 

require  0.5  m./]  00  CaCl2  ..  165 

2.2  c.c.  m./2  KC1  in  100  c.c.  H20 

require  0.3  m./lOO  CaCl2  . . 366 

2.75  c.c.  m./2  KC1  in  100  c.c.  H20 

require  1.0  m./lOO  CaCl2  . . 137.5 

3.3  c.c.  m./2  KC1  in  100  c.c.  H20 

require  1.6  m./lOO  CaCl2  . . 103 


CaCl2  acts  upon  KC1  similarly  as  does 
NaCl,  but  it  acts  more  powerfully;  i.  e.,  the 


coefficient  of  antagonization,  KCl/CaCl2, 
is  several  hundred  or  a thousand  times  as 
great  as  that  of  KCl/NaCl,  as  Table  V. 
shows. 

The  coefficients  are  not  as  regular  as  in 
the  case  of  antagonization  of  KC1  by'NaCl. 
This  is  due  to  the  fact  that  the  minimal 
value  of  CaCl2  at  which  it  renders  the  KC1 
harmless  can  not  be  determined  as  sharply 
as  the  limit  for  NaCl.  Why  is  less  CaCl2 
required  than  NaCl  ? We  can  only  answer 
with  a suggestion  first  offered  by  T.  B. 
Robertson,  namely,  that  CaCl2  produces  its 
protective  effect  through  the  formation  of 
a comparatively  insoluble  compound  (in 
this  case  on  the  gills  or  the  rest  of  the  sur- 
face of  the  animal)  while  NaCl  acts 
through  the  formation  of  a compound 
which  is  more  soluble.  This  view  is  cor- 
roborated by  the  observation  which  we 
made,  that  Sr  is  just  as  effective  to  antagon- 
ize KC1  as  CaCl2,  but  that  Mg  is  much  less 
efficient.  This  would  correspond  with  the 
well-known  fact  that  many  strontium  salts 
are  just  as  insoluble,  if  not  more  insoluble, 
than  the  calcium  salts,  while  the  magne- 
sium salts  are  often  incomparably  more 
soluble,  for  instance  in  the  case  of  the  sul- 
phates. BaCl2  antagonizes  KC1  also  pow- 
erfully, but,  probably,/ in  consequence  of 
the  fact  that  the  substances  formed  at  the 
surface  of  the  animal  or  the  gills,  diffuse 
slowly  into  the  cells,  the  fish  do  not  remain 
alive  as  long  if  Ba  is  used  as  if  the  more 
harmless  Ca  and  Sr  are  used. 

It  is  very  remarkable  that  CaCl2  renders 
harmless  any  given  concentration  of  KC1 
below  6.6  c.c.  m./2  KC1  in  100  c.c.  of  the 
solution,  but  not  above  this  limit.  This 
limit  is  exactly  the  same  which  we  found  in 
the  case  of  antagonization  of  KC]  by  NaCl 
Even  the  combination  of  NaCl  and  CaCl2 
does  not  permit  us  to  render  harmless  more 
than  6.6  c.c.  m./2  KC1  in  100  c.c.  of  the 
solution. 


10 


SCIENCE 


If  we  try  to  render  NaCl  harmless  by 
KC1  and  CaCl2  we  find  that  CaCl2  can 
antagonize  even  a 6/8  m.  and  a 7/8  m.  so- 
lution of  NaCl,  while  KC1  ceases  to  show 
any  antagonistic  effect  if  the  NaCl  solution 
exceeds  m./2  or  5/8  m. 

Experiments  with  pure  CaCl2  solutions 
give  the  result  that  this  substance  is  harm- 
less in  a solution  of  that  concentration  in 
which  this  salt  is  contained  in  the  sea- 
water. Fundulus  can  live  indefinitely  in 
a solution  of  1.5  c.c.  m./2  CaCl2  in  100  c.c. 
Botanists  have  also  found  that  weak  solu- 
tions of  CaCl2  are  comparatively  little 
toxic.  This  gives  us  the  impression  that 
the  effect  upon  the  surface  film  of  proto- 
plasm produced  by  CaCl2  is  especially  im- 
portant for  the  protection  of  the  proto- 
plasm. This  conclusion  receives  an  indirect 
support  by  the  well-known  experiments  of 
Herbst,  who  found  that  in  sea-water  de- 
prived of  calcium  the  segmentation  cells  of 
a sea-urchin  embryo  fall  apart  through  the 
disintegration  or  liquefaction  of  a film 
which  surrounds  the  embryo  and  keeps  the 
cells  together.  If  such  eggs  are  brought 
back  into  solution  containing  calcium  the 
film  is  restored  and  the  cells  come  into  close 
contact  again. 

It  is  therefore  not  impossible  that  the 
mechanism  of  the  antagonism  between  KC1 
and  NaCl  is  similar  to  that  found  between 
NaCl  and  ZnSo4.  It  seems  only  due  to  the 
high  concentration  of  the  NaCl  in  the  sea- 
water and  in  the  blood  that,  in  addition  to 
KC1  and  NaCl,  CaCl2  is  needed.  But  the 
case  is  not  so  unequivocal  as  the  previously 
mentioned  cases  of  antagonism  between 
only  two  electrolytes. 

VIII 

It  is  necessary  for  our  understanding  of 
the  life-preserving  action  of  salts  that  we 
do  not  depend  merely  on  conclusions  drawn 
from  experiments,  but  that  we  must  be  able 


to  see  directly  in  which  way  abnormal  salt 
solutions  cause  the  death  of  the  cell.  Such 
an  opportunity  is  offered  us  through  the 
observation  of  the  eggs  of  the  sea-urchin. 
If  we  put  the  fertilized  eggs  of  the  sea- 
urchin  into  an  abnormal  salt  solution,  a de- 
struction of  the  cell  gradually  takes  place. 
The  destruction,  as  a rule,  begins  on  the 
surface  of  the  protoplasm,  and  consists 
very  often  in  the  formation  and  falling  off 
of  small  granules  or  droplets.  This  process 
gradually  continues  from  the  periphery 
towards  the  center  until  the  whole  egg  is 
disintegrated.  For  different  salt  solutions 
the  picture  of  the  disintegration  is  a little 
different,  but  sufficiently  characteristic  for 
a given  solution,  so  that  if  one  become  fa- 
miliar with  these  pictures,  one  is  able  to 
diagnose  to  some  extent  the  nature  of  the 
solution  from  the  way  in  which  the  cell  dis- 
integrates. 

This  process  of  disintegration  can  be  ob- 
served if  the  eggs  are  put  into  a pure  solu- 
tion of  sodium  chloride  or  in  a mixture  of 
sodium  chloride  and  calcium  chloride,  or  in 
a mixture  of  sodium  chloride  and  potas- 
sium chloride.  If,  however,  all  three  salts 
are  used  in  the  proportion  in  which  they 
occur  in  the  sea-water  no  disintegration 
takes  place  and  the  surface  of  the  egg  re- 
mains perfectly  smooth  and  normal.  One 
gains  the  impression  as  if  the  protoplasm 
of  the  egg  were  held  together  by  a continu- 
ous surface  film  of  a definite  texture.  If 
we  put  the  egg  into  an  abnormal  solution 
this  surface  film  is  modified  and  changed, 
and  the  change  of  the  surface  film  is  often 
followed  by  a gradual  process  of  disinte- 
gration of  the  rest  of  the  cell. 

These  observations  on  the  sea-urchin 
egg,  therefore,  suggest  the  possibility  that 
the  combination  of  the  three  salts  in  their 
definite  proportion  and  concentration  has 
the  function  of  forming  a surface  film  of 
a definite  structure  or  texture,  around  the 


SCIENCE 


11 


protoplasm  of  each  cell,  by  which  the  proto- 
plasm is  kept  together,  protected  against 
and  separated  from  the  surrounding  media. 

The  previously  mentioned  observation  of 
Herbst  again  shows  the  important  role  of 
calcium  in  this  process. 

IX 

The  objection  might  be  raised  that  the 
beneficial  action  of  the  three  salts  could 
only  be  proved  on  marine  anipials  or  on 
tissues  of  higher  animals,  which  are  said 
to  be  “ adapted’ ’ to  a mixture  of  NaCl, 
KC1  and  CaCl2  in  definite  proportions. 
Experiments  on  fresh-water  organisms, 
for  which  “ adaptation’ * to  a mixture  of 
NaCl,  KC1  and  CaCl2  in  these  definite  pro- 
portions can  not  be  claimed,  show  that  this 
objection  is  not  valid.  Ostwald  worked 
with  fresh-water  crustaceans  which  he  put 
into  mixtures  of  various  salts.  It  was 
found  that  these  animals  live  longer  in  a 
mixture  of  NaCl  + KC1  + CaCl2  than  in  a 
solution  of  NaCl,  or  NaCl  + KC1,  or 
NaCl  + CaCl2  of  the  saipe  osmotic  pres- 
sure. 

Osterhout  was  able  to  show  that  the  spores 
of  a certain  variety  of  Vaucheria  die  in  a 
pure  3/32  m.  solution  of  NaCl  in  10  to  20 
minutes,  while  they  live  in  100  c.c.  3/32  m. 
NaCl  + 1 c.c.  3/32  CaCl2  2 to  4 weeks, 
and  in  100  c.c.  3/32  m.  NaCl  + 1 c.c. 
3/32  m.  CaCl2  + 2.2  c.c.  3/32  m.  KC1  6 to 
8 weeks.  The  reaction  of  the  solution  was 
strictly  neutral  and  the  NaCl  the  purest 
obtainable.  The  results  remained  the  same 
after  the  NaCl  had  been  recrystallized  six 
times.  Experiments  with  Spirogyra  gave 
a similar  result.  The  solutions  were  all 
3/32  m.  In  NaCl  the  Spirogyra  died  in 
18  hours ; in  NaCl  + KC1  in  two  days ; in 
NaCl  + KC1  + CaCl2  they  lived  65  days. 
Osterhout  caused  wheat  grains  to  develop 
in  such  solutions  and  measured  the  total 
length  of  the  roots  formed. 


Total  Length  of 

Nature  of  the  Solution  Roots  after  40  Days 

H20  740  mm. 

100  c.c.  3/25  NaCl  59  mm. 

1 00  c.c.  3/25  NaCl+2.0  3/25  CaCL,  254  mm. 

100  c.c.  3/25  NaCl+2.0  3/25  CaCl2 

+ 2.2  3/25  m.  KC1  324  mm. 

These  cases,  to  which  many  other  similar 
observations  might  be  added,  prove  that 
the  life-preserving  effect  of  the  combina- 
tion of  NaCl  + KC1  -j-  CaCl2  in  definite 
proportions  is  not  due  to  the  fact  that  or- 
ganisms are  “adapted”  to  this  mixture  but 
to  a specific  protective  effect  of  the  combi- 
nation of  the  three  salts  upon  the  cells. 

x 

It  seems,  therefore,  to  be  a general  fact 
that  wherever  tissues  or  animals  require  a 
medium  of  a comparatively  high  osmotic 
pressure — like  our  tissues — their  life  lasts 
much  longer  in  a mixture  of  NaCl  + KCl  + 
CaCl2  in  the  proportion  in  which  these 
salts  exist  in  the  blood  and  in  the  ocean, 
than  in  any  other  osmotic  solution,  even  a 
pure  solution  of  NaCl.  But  the  reader  has 
noticed  that  there  are  considerable  differ- 
ences in  the  resistance  of  various  organ- 
isms to  abnormal  solutions.  While  marine 
Gammarus  die  in  half  an  hour  in  an  iso- 
tonic solution  of  NaCl  or  cane  sugar,  red 
blood  corpuscles  or  even  the  muscle  of  a 
frog  can  be  kept  for  a day  or  longer  in 
such  a solution  (of  course  even  the  muscle 
of  a frog  lives  longer  if  the  NaCl  solution 
contains  in  addition  KC1  or  CaCl2).  What 
causes  this  difference? 

Six  years  ago  I found  that  the  unfertil- 
ized eggs  of  the  sea-urchin  ( Strongylocen - 
trotus  purpuratus)  can  keep  alive  and  re- 
main apparently  intact  in  a pure  neutral 
solution  of  CaCl2  or  of  NaCl  for  several 
days  at  a temperature  of  15°,  while  the 
fertilized  eggs  of  the  same  female  are 
killed  in  a pure  neutral  solution  of  CaCl2 
in  a few  hours.  The  same  difference  is 


12 


SCIENCE 


found  for  other  salts  also.  What  causes 
this  difference?  Several  authors,  Lillie, 
McClendon  and  Lyon,  have  suggested  that 
it  is  due  to  the  fact  that  the  fertilized  egg 
is  more  permeable  to  salts  than  the  unfer- 
tilized egg.  But  the  recent  experiments  by 
Warburg,  which  were  confirmed  and  ampli- 
fied by  Harvey  make  it  doubtful  whether 
the  salts  which  are  not  soluble  in  fats  can 
enter  the  fertilized  egg  at  all.  I believe 
that  the  explanation  of  the  difference  is 
much  more  simple.  The  unfertilized  egg  is 
surrounded  by  a cortical  layer  and  this 
layer  is  destroyed  or  modified  in  the  proc- 
ess of  fertilization.  One  result  of  this 
modification  is  the  formation  of  the  fertili- 
zation membrane,  for  which  I have  been 
able  to  show  that  it  is  readily  permeable 
for  salts.  As  long  as  the  cortical  layer  of 
the  unfertilized  egg  is  intact,  it  prevents 
the  surrounding  salt  solution  from  coming 
in  contact  with  the  protoplasm  or  at  least 
it  retards  this  process.  If,  however,  the 
cortical  layer  is  destroyed  by  fertilization 
the  surrounding  salt  solution  comes  directly 
in  contact  with  the  protoplasm  and  if  the 
solution  is  abnormal  it  can  cause  the  dis- 
integration of  the  surface  layer  of  the 
protoplasm. 

I am  inclined  to  believe  that  differences 
in  the  resisting  power  of  various  cells  or 
organisms  to  abnormal  salt  solutions  are 
primarily  due  to  differences  in  the  consti- 
tution of  the  protective  envelopes  of  the 
animals  or  the  cells.  Microorganisms 
which  can  live  in  strong  organic  acids  or 
salt  solutions  of  a high  concentration  prob- 
ably possess  a surface  layer  which  shuts  off 
their  protoplasm  from  contact  with  the  so- 
lution. For  the  protoplasm  of  muscle  the 
rather  tough  sarcolemma  forms  not  an 
absolute  but  nevertheless  an  effective  wall 
against  the  surrounding  solution. 

But  aside  from  differences  of  this  kind 
there  are  other  conditions  which  influence 


the  degree  of  resistance  of  cells  to  various 
solutions.  I have  found  that  the  fertilized 
eggs  of  the  sea-urchin  will  live  longer  in 
abnormal  salt  solutions  if  the  oxidations  in 
the  egg  are  stopped,  either  by  the  with- 
drawal of  oxygen  or  the  addition  of  KCN 
or  NaCN.  Warburg  and  Meyerhof  have 
drawn  the  conclusion  that  in  a pure  NaCl 
solution  the  rate  of  oxidations  of  the  egg  of 
Strongylocentrotus  is  increased  and  that 
it  is  this  increase  in  the  rate  of  oxidations 
which  kills  the  eggs.  But  this  increase  of 
oxidations  can  not  be  observed  in  the  eggs 
of  Arbacia  when  they  are  put  into  a pure 
NaCl  solution  and,  moreover,  lack  of 
oxygen  prolongs  the  life  of  the  fertilized 
egg  just  as  well  in  solutions  of  NaCl  + 
CaCl2  or  of  NaCl  + BaCl2,  for  which  salts 
these  authors  do  not  claim  that  they  can 
raise  the  rate  of  oxidations  of  the  egg.  I 
am  inclined  to  believe  that  in  the  process 
during  or  preceding  cell  division,  besides 
phenomena  of  streaming  inside  the  cell, 
changes  in  the  surface  film  of  the  proto- 
plasm occur,  whereby  this  film  is  more 
easily  injured  by  the  salts.  If  we  suppress 
the  oxidations  we  suppress  also  the  proc- 
esses leading  to  cell  division  and  thereby 
retard  the  deleterious  action  of  the  ab- 
normal salt  solution  upon  the  surface  layer 
of  the  protoplasm  of  the  egg. 

XI 

If  we  now  raise  the  question  as  to  why 
salts  are  necessary  for  the  preservation  of 
the  life  of  the  cell  we  can  point  to  a num- 
ber of  cases  in  which  this  answer  seems 
clear.  Each  cell  may  be  considered  a chem- 
ical factory,  in  which  the  work  can  only 
go  on  in  the  proper  way,  if  the  diffusion 
of  substances  through  the  cell  wall  is 
restricted.  This  diffusion  depends  on  the 
nature  of  the  surface  layer  of  the  cell. 
Overton  and  others  assume  that  this  layer 
consists  of  a continuous  membrane  of  fat 


SCIENCE 


13 


or  lipoids.  This  assumption  is  not  com- 
patible with  two  facts,  namely  that  water 
diffuses  very  rapidly  into  the  cell,  and 
second,  that  life  depends  upon  an  exchange 
of  water-soluble  and  not  of  fat-soluble 
substances  between  the  cells  and  the  sur- 
rounding liquid.  The  above  mentioned 
facts  of  the  antagonism  between  acids  and 
salts  suggest  the  idea  that  the  surface  film 
of  cells  consists  exclusively  or  essentially 
of  certain  proteins. 

The  experiments  mentioned  in  this  paper 
indicate  that  the  role  of  salts  in  the  preser- 
vation of  life  consists  in  the  “ tanning  ” 
effect  which  they  have  upon  the  surface 
films  of  the  cells,  whereby  these  films  ac- 
quire those  physical  qualities  of  durability 
and  comparative  impermeability,  without 
which  the  cell  cannot  exist. 

On  this  assumption  we  can  understand 
that  neutral  salts  should  be  necessary  for 


the  preservation  of  life  although  they  do 
not  furnish  energy. 

As  far  as  the  dynamical  effects  of  salts 
are  concerned  it  is  not  impossible  that 
some  of  them  belong  also  to  the  type  of  those 
mentioned  in  this  paper.  The  fact  that 
the  addition  of  calcium  to  an  NaCl  solu- 
tion prevents  the  twitchings  of  the  muscle, 
which  occur  in  the  pure  NaCl  solution, 
suggests  the  possibility  that  the  CaCl2 
merely  prevents  or  retards  the  diffusion  of 
NaCl  through  the  sarcolemma.  But  other 
effects  of  salts,  e.  g.,  the  apparent  depend- 
ence of  contractility  of  the  muscle  upon 
the  presence  of  NaCl ; or  the  role  of  P04  in 
the  nucleus,  do  not  find  their  explanation 
in  the  facts  discussed  in  this  paper. 

Jacques  Loeb 

Rockefeller  Institute 
for  Medical  Research 


5 


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